Rho GTPases comprise a branch of the Ras superfamily of small GTPases. They play a key role in the modulation of a wide array of cellular processes including cell migration, cell polarization, membrane trafficking, cytoskeleton arrangements, proliferation, apoptosis, and transcriptional regulation. (Etienne-Manneville, S. et al (2002). Nature 420,629-635; Boettner, B. et al. (2002). Gene 286, 155-174.) Hence, Rho GTPases have been implicated in the pathogenesis of various human diseases including cardiovascular diseases and cancer (Hall, A. Science 1998, 279, 509-514; Wennerberg, K., and Der, C. J. (2004) J. Cell Sci. 117, 1301-1312.; Ridley, A. J. (2006) Trends Cell Biol. 16, 522-529).
The Rho family is comprised of 22 genes encoding at least 25 proteins in humans including Rac. Rho family members bind GTP and transition between an inactive GDP-bound and an active GTP-bound state. In doing so, many of the Rho family members exhibit a GTPase activity when in their active state. This cycling between states is regulated by: guanine nucleotide exchange factors (GEFs); the GTPase activating proteins (GAPs); and GDP dissociation inhibitors (GDIs) which act as negative regulators. (Malumbres, M. et al (2003) Nat. Rev. Cancer 3, 459-465). In quiescent cells, Rho GTPases are predominantly present in an inactive GDP bound state whereas upon growth stimulation, GEFs are activated and subsequently stimulate the guanine nucleotide exchange activity to promote formation of the active GTP bound Rho. When bound to GTP, active Rho GTPases interact with downstream effectors including protein kinases and other proteins with adaptor functions. The intrinsic GTP hydrolysis functionality of Rho GTPases is later stimulated by the Rho specific GTPase activating protein. This returns the Rho protein to its inactive state. Rac-specific RhoGEFs include Tiam1 and Trio (Gao, Y. et al. (2004). Proc. Natl. Acad. Sci. USA 101, 7618-7623.)
The Rac subfamily has also been linked to cellular transformation and hence, the aberrant activity of Rho GTPases is associated with cancer. They play an essential role in transformation caused by Ras and other oncogenes. The Rac1b splice variant of Rac1 has been shown to be constitutively active and transforming; its overexpression has been observed in both breast and colon cancers (Qiu, R. G., et al. (1995) Nature 374, 457-459; Khosravi-Far, R., et al (1995) Mol. Cell. Biol. 15, 6443-6453; Renshaw, M. W. et al (1996) Curr. Biol. 6, 76-83; Ferraro, D., et al. (2006) Oncogene 25, 3689-3698). Rac3 mutants, for example, have been noted in brain tumors and both Rac1 and Rac3 have been linked to glioblastoma invasion (Hwang, S. L. et al (2005) J. Clin. Neurosci. 12, 571-574).
In malignant cells, aberrant Rho GTPase activity results from changes in the expression of Rho GTPases or the perturbed function of either GEFs or GAPs which regulate the function of Rho. (Karnoub, A. E. et al (2004). Breast Cancer Res. Treat. 84, 61-71.) Due to the evidence of Rho involvement in cell transformation, Rho GTPases are probable targets for anti-cancer therapies. Compounds that inhibit GEF interaction with their respective Rho family members would be useful inhibitors of Rho activity and exhibit great specificity. To date, small molecule NSC23766 (i.e., N6-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride) has been identified as binding to Rac1 and preventing its activation by Rac-specific RhoGEFs. Some GEF activity, however, was not blocked.
Chronic myelogenous leukemia (CML) is a malignant disease characterized by expression of p210-BCR-ABL, the product of the Philadelphia chromosome. Also known as chronic granulocytic leukemia (CGL), it is a cancer of the white blood cells and is characterized by the increased and upregulated growth of mainly myeloid cells in the bone marrow and the accumulation of these cells in the blood. The deficiency of the Rho GTPases Rac1 and Rac2 in a murine model has shown a significant reduction of p210-BCR-ABL-mediated proliferation. Rac has also been shown to play a role in other types of leukemias such as MLL-mediated acute leukemia. (Mizukawa B. et al., Blood 2011; 118:5235-45). The above evidence has strongly suggested Rac as a potential target for leukemia therapy. (E K Thomas et al, Leukemia 22, 898-904, May 2008).